Protein specific for cardiac and skeletal muscle

ABSTRACT

The present invention relates to a muscle-specific protein, Ozz, and nucleic acids encoding the protein, that regulates development and function of muscle cells. The invention further relates to muscle-specific regulated expression of the protein, and of heterologous genes under control of the same regulatory sequences. In a specific example, a murine Ozz protein of 285 amino acids is preferentially expressed by a 1.0 kb mRNA in heart and skeletal muscle. This protein shares significant homology with neuralized proteins, and associates with a number of muscle proteins, including β-catenin.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. §119 based upon U.S.Provisional Application Ser. No. 60/131,814 filed Apr. 29, 1999, theentire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a muscle-specific protein, and nucleicacids encoding the protein, that regulates development and function ofmuscle cells. The invention further relates to muscle-specific regulatedexpression of the protein, and of heterologous genes under control ofthe same regulatory sequences.

BACKGROUND OF THE INVENTION

The human and mouse protective protein/cathepsin A (PPCA) genes arehighly homologous, both in sequence and organization. Their expressionin mammalian tissues is ubiquitous but differential. The mouse gene istranscribed from two promoters, giving rise to two mRNAs which differ insize and tissue distribution. The less abundant, larger transcript of2.0 kb is present only in a few tissues and is transcribed from atissue-specific promoter, upstream of the constitutive promoter presentin both the human and mouse genes. Northern blot hybridizationsdemonstrated that these two mRNAs differed only in their 5′UTRs.

PPCA is a lysosomal carboxypeptidase that is deficient in the humanlysosomal storage disease galactosialidosis (reviewed in d'Azzo et al.,In The Metabolic and Molecular Bases of Inherited Disease, C. Scriver etal. (eds.), New York: McGraw-Hill Publishing Co., pp. 2825-38, 1995).The human and mouse PPCA cDNAs are 85% homologous in their codingregions and 72% identical in the 3′ untranslated regions (Galjart, etal., Cell, 54:755-764, 1988; Galjart, et al., J. Biol. Chem.,265:4678-84, 1990). The genomic organization and structure of the twoPPCA genes are also conserved. The human gene maps to chromosome 20q13.1and the mouse to the syntenic region of chromosome 2-H4 (Wiegant, etal., Genomics, 10:345-349, 1991; Williamson, et al., Genomics,22:240-242, 1994; Shimmoto, et al., Biochem. Biophys. Res. Comm.,220:802-806, 1996; Rottier and d'Azzo, DNA Cell Biol., 16:599-610,1997). Shimmoto, et al. showed that the last exon of the human gene,exon XV, partially overlaps over a stretch of 58 nucleotides (nt) withthe gene encoding the phospholipid transfer protein (PLTP; Shimmoto, etal., supra). Similarly, the murine PPCA and PLTP genes are in closevicinity and probably overlap as well. Whether these two genes sharecommon regulatory elements is unknown at the moment.

SUMMARY OF THE INVENTION

The present invention provides a novel protein, termed Ozz, which isinvolved in development and function of muscle. Thus, in one aspect, anisolated and, preferably, purified, Ozz protein is provided. In specificembodiments, the protein is a human Ozz or a murine Ozz.

Also provided are fragments, analogs, and derivatives of Ozz, which arecharacterized by the ability to bind a protein selected from the groupconsisting of β-catenin, myosin, c-Nap, and Alix.

In a further embodiment, a polypeptide fragment of Ozz protein isprovided. The fragment of Ozz may have a property of about 40% sequenceidentity to a duplicated neuralized homology repeat of neuralizedprotein of Drosphila; or a polypeptide comprising a stretch of about 30amino acids at the C-terminus homologous to two regions of neutralizedproteins; or a peptide comprising an amino acid sequence selected formthe group consisting of GTRATR (SEQ ID NO:19), GVCFSR (SEQ ID NO:20),GQPEA (SEQ ID NO:21), and KGLKDFCKY (SEQ ID NO:22); or specific bindingactivity with an anti-Ozz antibody.

The invention further provides an isolated oligonucleotide encoding Ozzprotein. Further provided is a vector comprising a nucleic acid encodinga polypeptide fragment of Ozz, including full length Ozz, operativelyassociated with an expression control sequence, wherein the polypeptidehas the ability to bind a β-catenin, myosin, c-Nap, or Alix protein, aswell as a cell comprising such a vector, or a non-human animaltransformed with such a vector.

In a further embodiment, the invention provides an isolated nucleic acidof at least ten bases, preferably of at least about 17 to about 20bases, that hybridizes under stringent conditions with a nucleic acidhaving a nucleotide sequence as depicted in SEQ ID NO:1 or 3, with theproviso that the nucleic acid is not a PPCA exon I.

The invention further provides an Ozz muscle-specific promoter, whichprovides for targeted, tissue-specific expression.

A further aspect of the invention relates to an antibody thatspecifically binds to Ozz protein, and related methods for detecting anOzz protein comprising detecting binding of the antibody to a protein ina sample suspected of containing an Ozz protein.

Alternatively, expression of Ozz can be detected by detecting mRNAencoding Ozz in a sample from a cell suspected of expressing Ozz.

In a further embodiment, the discovery of Ozz provides a method fordetecting damage to muscle tissue comprising detecting an increase inthe level of Ozz protein in a blood or a blood fraction, wherein thepresence of an increase in the level of Ozz in blood or a blood fractionindicates damage to muscle tissue.

Another embodiment of the invention relates to a method of detecting adisease associated with a defect in Ozz expression comprising detectingan abnormal amount or localization pattern of Ozz in muscle cells from asubject.

These and further aspects of the invention are more fully disclosed inthe drawings, description, and examples below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic drawing of genetic structure of the PPCA and Ozzgenes. The Ozz gene is enlarged; the mRNA structure is shown below it.

FIGS. 2A and B. (A) Murine Ozz cDNA sequence (SEQ ID NO:1) and (B) humanOzz cDNA sequence (SEQ ID NO:3). The ATG translation start site and TGAtermination site are indicated with bold text.

FIG. 3. Schematic representation of the human and mouse 5′ genomicregions (top), and sequence comparison of their 5′ UTRs and adjacentpromoter regions (bottom; the human distal promoter is SEQ ID NO:13; themurine distal promoter is SEQ ID NO:14; the human proximal promoter isSEQ ID NOS:15 and 16; and the murine proximal promoter is SEQ ID NOS:17and 18). Percentage of homology is indicated. The 5′ UTR exons are inboldface and putative transcription factor binding sites are indicated.Two imperfect CAAT boxes are underlined in the murine distal promoter(bottom, left; SEQ ID NO:14). The transcriptional start sites, found byusing RNase protection assays, are marked with an arrow above (human) orbelow (mouse) the sequence.

FIG. 4. Amino acid sequence of mouse Ozz protein (SEQ ID NO:2) and humanOzz protein (SEQ ID NO:4). Putative phosphorylation sites for casein II(T44, T86, and S100; light shading), protein kinase C (T184, S216, S235,and S238; dark shading), and tyrosine kinase (Y284; medium shading); andSOCS box (residues 249-285; darker shading), and BC box (residues250-259; lighter shading) consensus domains are shown.

FIG. 5. Schematic representation of the Ozz and neuralized proteins,showing regions of homology between the two primary sequences.

FIG. 6. Sequence alignment between Ozz protein (SEQ ID NOS:5, 7, 9, and11) and D. virilis neuralized protein (SEQ ID NOS:6, 8, 10, and 12).

FIG. 7. Immunoprecipitation of Ozz protein in C₂C₁₂ and COS transfectedcells, using anti-Ozz antibodies.

FIG. 8. Human multi-tissue Northern blot using full-length mouse OzzcDNA as a probe.

DETAILED DESCRIPTION OF THE INVENTION

Analysis of the 5′ regions of the human and mouse PPCA genes led to theidentification of their minimal promoter, which bears characteristics ofhousekeeping gene promoters and drives the transcription of anubiquitously expressed mRNA. In addition, an alternative upstreampromoter, present only in the mouse gene, that controls expression of alarger transcript of 2 kb present only in some tissues was found(Rottier and d'Azzo, DNA Cell Biol., 16:599-610, 1997). Functionalanalysis of the second PPCA transcript in mouse tissues brought thediscovery of a new transcriptional unit, overlapping with exon Ia of themouse PPCA gene. Thus, the present invention is based, in part, on theisolation and characterization of the corresponding cDNA and encodedprotein, which has been named Ozz.

The primary structure of Ozz shows homology to the product of adevelopmental gene of Drosophila, called neuralized (neu), also found inC. elegans and humans. The Drosophila neu is a member of the neurogenicgene family that determine the cell fate in the developing centralnervous system of the fly embryo (Corbin, et al, Cell, 67:311-23, 1991;Martin-Bermundo, et al., Development, 121:219-24, 1995; Hartenstein, etal, Development, 116:203-20, 1992). Absence of neu in the embryo resultsin an excess of neuroblasts, suggesting that neuralized plays a role indetermining cell fate in the neurogenic region of the embryo. Theneuralized protein contains four domains: a nuclear localization signal,a homeodomain similarity, a helix-turn-helix motif, a zinc-fingerregion, as well as a potential DNA binding domain, the RING zinc-fingerdomain (Price, et al., EMBO J., 12:2411-18, 1993). In addition,neuralized includes a stretch of 100 amino acids which is repeated twicein the Drosophila, human and C. elegans proteins, and is called NHR(Neuralized Homologous Region) (Nakamura, et al., Oncogene, 16:1009-19,1998). The NHR domain is present in the N-terminal region of the Ozzprotein. Ozz mRNA and protein are preferentially expressed in embryonaland adult muscle tissues. Ozz is likely to be involved in muscledifferentiation.

General Definitions

As used herein, the term “isolated” means that the referenced materialis removed from the environment in which it is normally found. Thus, anisolated biological material can be free of cellular components, i.e.,components of the cells in which the material is found or produced. Inthe case of nucleic acid molecules, an isolated nucleic acid includes aPCR product, an isolated mRNA, a cDNA, or a restriction fragment. Inanother embodiment, an isolated nucleic acid is preferably excised fromthe chromosome in which it may be found, and more preferably is nolonger joined to non-regulatory, non-coding regions, or to other genes,located upstream or downstream of the gene contained by the isolatednucleic acid molecule when found in the chromosome. In yet anotherembodiment, the isolated nucleic acid lacks one or more introns.Isolated nucleic acid molecules include sequences inserted intoplasmids, cosmids, artificial chromosomes, and the like. Thus, in aspecific embodiment, a recombinant nucleic acid is an isolated nucleicacid. An isolated protein may be associated with other proteins ornucleic acids, or both, with which it associates in the cell, or withcellular membranes if it is a membrane-associated protein. An isolatedorganelle, cell, or tissue is removed from the anatomical site in whichit is found in an organism. An isolated material may be, but need notbe, purified.

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate the presence ofunrelated materials, i.e., contaminants, including native materials fromwhich the material is obtained. For example, a purified protein ispreferably substantially free of other proteins or nucleic acids withwhich it is associated in a cell; a purified nucleic acid molecule ispreferably substantially free of proteins or other unrelated nucleicacid molecules with which it can be found within a cell. As used herein,the term “substantially free” is used operationally, in the context ofanalytical testing of the material. Preferably, purified materialsubstantially free of contaminants is at least 50% pure; morepreferably, at least 90% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art.

Methods for purification are well-known in the art. For example, nucleicacids can be purified by precipitation, chromatography (includingpreparative solid phase chromatography, oligonucleotide hybridization,and triple helix chromatography), ultracentrifugation, and other means.Polypeptides and proteins can be purified by various methods including,without limitation, preparative disc-gel electrophoresis, isoelectricfocusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange andpartition chromatography, precipitation and salting-out chromatography,extraction, and countercurrent distribution. For some purposes, it ispreferable to produce the polypeptide in a recombinant system in whichthe protein contains an additional sequence tag that facilitatespurification, such as, but not limited to, a polyhistidine sequence, ora sequence that specifically binds to an antibody, such as FLAG and GST.The polypeptide can then be purified from a crude lysate of the hostcell by chromatography on an appropriate solid-phase matrix.Alternatively, antibodies produced against the protein or againstpeptides derived therefrom can be used as purification reagents. Cellscan be purified by various techniques, including centrifugation, matrixseparation (e.g., nylon wool separation), panning and otherimmunoselection techniques, depletion (e.g., complement depletion ofcontaminating cells), and cell sorting (e.g., fluorescence activatedcell sorting [FACS]). Other purification methods are possible. Apurified material may contain less than about 50%, preferably less thanabout 75%, and most preferably less than about 90%, of the cellularcomponents with which it was originally associated. The “substantiallypure” indicates the highest degree of purity which can be achieved usingconventional purification techniques known in the art.

In a specific embodiment, the term “about” or “approximately” meanswithin 20%, preferably within 10%, and more preferably within 5% of agiven value or range.

A “sample” as used herein refers to a biological material which can betested for the presence of Ozz protein or Ozz nucleic acids. Suchsamples can be obtained from animal subjects, such as humans andnon-human animals, and include tissue, especially muscle, biopsies,blood and blood products (plasma and serum, e.g., for released Ozzprotein; or blood cells, particularly nucleated cells, for possibledetection of protein or nucleic acids); plural effusions; cerebrospinalfluid (CSF); ascites fluid; and cell culture.

Non-human animals include, without limitation, laboratory animals suchas mice, rats, rabbits, hamsters, guinea pigs, etc.; domestic animalssuch as dogs and cats; and, farm animals such as sheep, goats, pigs,horses, and cows.

The use of italics indicates a nucleic acid molecule (e.g., Ozz cDNA,gene, etc.); normal text indicates the polypeptide or protein.

Thus, the present invention advantageously provides Ozz protein,including fragments, derivatives, and analogs of Ozz; Ozz nucleic acids,including oligonucleotide primers and probes, and Ozz regulatorysequences; Ozz-specific antibodies; and related methods of using thesematerials to detect the presence of Ozz proteins or nucleic acids, Ozzbinding partners, and in screens for agonists and antagonists of Ozz.The following sections of the application, which are delineated byheadings (in bold) and sub-headings (in bold italics), which cover theseaspects of the invention, are provided for clarity, and not by way oflimitation.

Ozz

Ozz protein, as defined herein, refers to a polypeptide having about 285amino acids. In a specific embodiment, human Ozz has 285 amino acids. Inanother specific embodiment, murine Ozz also has 285 amino acids. Ozzcan have a calculated molecular weight of about 31.5 kilo-Daltons (kDa);when murine Ozz is expressed in a recombinant cell line, for example,the apparent molecular weight is about 29-30 kDa, as measured bySDS-polyacrylamide gel electrophoresis. Because they are highlyhomologous, human Ozz can be expected to have very similar properties.Indeed, human and murine Ozz share 90% sequence identity, and 92%sequence similarity. Thus, the term Ozz encompasses polypeptides havingabout 90% sequence identity or about 92% sequence similarity with SEQ IDNO:2 or 4 (murine or human Ozz). The N-terminal portion of Ozz hassignificant homology with Neuralized protein. In a specific embodiment,there is about 40% sequence identity between the N-terminal portion ofOzz and a duplicated repeat of Drosophila neuralized protein. Inaddition, there is a stretch of about 30 amino acids at the C-terminusof Ozz that shows homology to two regions of neuralized protein. In aspecific embodiment, the regions of homology of both the N-terminal andC-terminal regions of Ozz with Drosophila neuralized are shown in FIGS.5 and 6.

Ozz can be further characterized by a tissue-specific expressionpattern. Both Ozz mRNA and Ozz protein are only observed in heart andskeletal muscle, using routine assays (Northern analysis for mRNA andWestern analysis for protein). This tissue-specific expression patternhas been observed for both mice and humans. It has also been found to beexpressed in mice starting at embryonic day 12.5 (E12.5).

Ozz can also be characterized by the proteins to which it binds. Inspecific embodiments, using a yeast two-hybrid screen, Ozz was found toassociate with β-catenin, myosin, c-Nap, and Alix proteins. Furtherevidence of Ozz association with β-catenin was found byco-immunoprecipitation analysis. In another embodiment, Ozz binds to anOzz-specific antibody, e.g., as exemplified below.

In a specific embodiment, in order to develop the specific C-terminaland N-terminal Ozz antibodies, antibodies can be raised against the twohalves of Ozz protein. The two peptides are produced from two truncatedforms of mouse Ozz cDNA (corresponding to the nt 1-483 and nt 478-1036,respectively) fused with the GST coding sequence. The N terminus hasneuralized homology.

Ozz fragments, derivatives, and analogs can be characterized by one ormore of the characteristics of Ozz protein. For example, an Ozzfragment, also termed herein an Ozz peptide or polypeptide, can have anamino acid sequence corresponding to a homology region of neuralizedprotein, and in particular one of the fragments having SEQ ID NO:5, 7,9, or 11 (the homologous fragments of Ozz shown in FIG. 6). In addition,an Ozz peptide can have an amino acid sequence of the SOCS box havingthe sequence PSLQTLCRLVIQRSMVHRLAIDGLHLPKELKDFCKYE (SEQ ID NO:23), orthe amino acid sequence of a BC box having the sequence SLxxxCxxxI (SEQID NO:24). In another embodiment, an Ozz fragment comprises a putativephosphorylation site, e.g., a site for casein II (in a specificembodiment, such as site has a sequence as depicted in SEQ ID NO:19),protein kinase C (in a specific embodiment, such a site has a sequenceas depicted in SEQ ID NO:20), or tyrosine kinase (in a specificembodiment, such a site has a sequence as depicted in SEQ ID NO:22). Inyet another embodiment, an Ozz fragment can contain an alternativemodification site, for example a myristoylation site (in a specificembodiment, such a site has a sequence as depicted in SEQ ID NO:21).

Analogs and derivatives of Ozz of the invention have the same orhomologous characteristics of Ozz as set forth above. For example, atruncated form of Ozz can be provided. Such a truncated form includesOzz with a deletion. In a specific embodiment, the derivative isfunctionally active, i.e., capable of exhibiting one or more functionalactivities associated with a full-length, wild-type Ozz of theinvention. Such functions include binding β-catenin. Alternatively, anOzz chimeric fusion protein can be prepared in which the Ozz portion ofthe fusion protein has one or more characteristics of Ozz. Such fusionproteins include fusions of Ozz polypeptide with a marker polypeptide,such as FLAG, a histidine tag, or, as exemplified herein,glutathione-S-transferase (GST). Ozz can also be fused with a uniquephosphorylation site for labeling. In another embodiment, Ozz can beexpressed as a fusion with a bacterial protein, such as β-galactosidase.

Ozz analogs can be made by altering encoding nucleic acid sequences bysubstitutions, additions or deletions that provide for functionallysimilar molecules, i.e., molecules that perform one or more Ozzfunctions. In a specific embodiment, an analog of Ozz is asequence-conservative variant of Ozz. In another embodiment, an analogof Ozz is a function-conservative variant. In yet another embodiment, ananalog of Ozz is an allelic variant or a homologous variant from anotherspecies. In a specific embodiment, human and murine variants of Ozz aredescribed.

Ozz derivatives include, but are by no means limited to, phosphorylatedOzz, myristylated Ozz, methylated Ozz, and other Ozz proteins that arechemically modified. Ozz derivatives also include labeled variants,e.g., radio-labeled with iodine (or, as pointed out above, phosphorous);a detectable molecule, such as but by no means limited to biotin, achelating group complexed with a metal ion, a chromophore orfluorophore, a gold colloid, or a particle such as a latex bead; orattached to a water soluble polymer.

Chemical modification of biologically active component or components ofOzz may provide additional advantages under certain circumstances, suchas increasing the stability and circulation time of the component orcomponents and decreasing immunogenicity. See U.S. Pat. No. 4,179,337,Davis et al., issued Dec. 18, 1979. For a review, see Abuchowski et al.,in Enzymes as Drugs (J. S. Holcerberg and J. Roberts, eds. pp. 367-383(1981)). A review article describing protein modification and fusionproteins is Francis, 1992, Focus on Growth Factors 3:4-10, Mediscript:Mountview Court, Friern Barnet Lane, London N20, OLD, UK.

The chemical moieties suitable for derivatization may be selected fromamong water soluble polymers. The polymer selected should be watersoluble so that the component to which it is attached does notprecipitate in an aqueous environment, such as a physiologicalenvironment. Preferably, for therapeutic use of the end-productpreparation, the polymer will be pharmaceutically acceptable. Oneskilled in the art will be able to select the desired polymer based onsuch considerations as whether the polymer/component conjugate will beused therapeutically, and if so, the desired dosage, circulation time,resistance to proteolysis, and other considerations. For the presentcomponent or components, these may be ascertained using the assaysprovided herein.

The water soluble polymer may be selected from the group consisting of,for example, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols and polyvinyl alcohol. Polyethylene glycol propionaldenhyde mayadvantages in manufacturing due to its stability in water.

Cloning and Expression of Ozz

The present invention contemplates analysis and isolation of a geneencoding a functional or mutant Ozz, including a full length, ornaturally occurring form of Ozz, and any antigenic fragments thereoffrom any source, preferably human. It further contemplates expression offunctional or mutant Ozz protein for evaluation, diagnosis, or therapy.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. ÊPerbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Molecular Biology—Definitions

“Amplification” of DNA as used herein denotes the use of polymerasechain reaction (PCR) to increase the concentration of a particular DNAsequence within a mixture of DNA sequences. For a description of PCR seeSaiki et al., Science, 239:487, 1988.

“Chemical sequencing” of DNA denotes methods such as that of Maxam andGilbert (Maxam-Gilbert sequencing, Maxam and Gilbert, Proc. Natl. Acad.Sci. USA, 74:560, 1977), in which DNA is randomly cleaved usingindividual base-specific reactions.

“Enzymatic sequencing” of DNA denotes methods such as that of Sanger(Sanger et al., 1977, Proc. Natl. Acad. Sci. USA, 74:5463, 1977), inwhich a single-stranded DNA is copied and randomly terminated using DNApolymerase, including variations thereof well-known in the art.

As used herein, “sequence-specific oligonucleotides” refers to relatedsets of oligonucleotides that can be used to detect allelic variationsor mutations in the Ozz gene.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear (e.g., restrictionfragments) or circular DNA molecules, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A “polynucleotide” or “nucleotide sequence” is a series of nucleotidebases (also called “nucleotides”) in DNA and RNA, and means any chain oftwo or more nucleotides. A nucleotide sequence typically carries geneticinformation, including the information used by cellular machinery tomake proteins and enzymes. These terms include double or single strandedgenomic and cDNA, RNA, any synthetic and genetically manipulatedpolynucleotide, and both sense and anti-sense polynucleotide (althoughonly sense stands are being represented herein). This includes single-and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNAhybrids, as well as “protein nucleic acids” (PNA) formed by conjugatingbases to an amino acid backbone. This also includes nucleic acidscontaining modified bases, for example thio-uracil, thio-guanine andfluoro-uracil.

The polynucleotides herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, andinternucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters,phosphoroamidates, carbamates, etc.) and with charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.). Polynucleotides maycontain one or more additional covalently linked moieties, such as, forexample, proteins (e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, iron, oxidative metals,etc.), and alkylators. The polynucleotides may be derivatized byformation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the polynucleotides herein mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin, and the like.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, or used or manipulated in any way, for theproduction of a substance by the cell, for example the expression by thecell of a gene, a DNA or RNA sequence, a protein or an enzyme. Hostcells can further be used for screening or other assays, as describedinfra.

Proteins and enzymes are made in the host cell using instructions in DNAand RNA, according to the genetic code. Generally, a DNA sequence havinginstructions for a particular protein or enzyme is “transcribed” into acorresponding sequence of RNA. The RNA sequence in turn is “translated”into the sequence of amino acids which form the protein or enzyme. An“amino acid sequence” is any chain of two or more amino acids. Eachamino acid is represented in DNA or RNA by one or more triplets ofnucleotides. Each triplet forms a codon, corresponding to an amino acid.For example, the amino acid lysine (Lys) can be coded by the nucleotidetriplet or codon AAA or by the codon AAG. (The genetic code has someredundancy, also called degeneracy, meaning that most amino acids havemore than one corresponding codon.) Because the nucleotides in DNA andRNA sequences are read in groups of three for protein production, it isimportant to begin reading the sequence at the correct amino acid, sothat the correct triplets are read. The way that a nucleotide sequenceis grouped into codons is called the “reading frame.”

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme. A codingsequence for a protein may include a start codon (usually ATG) and astop codon.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more proteins or enzymes, and mayor may not include regulatory DNA sequences, such as promoter sequences,which determine for example the conditions under which the gene isexpressed. Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence. Other genes may function as regulators of structural genes oras regulators of DNA transcription.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” or “operatively associatedwith” of transcriptional and translational control sequences in a cellwhen RNA polymerase transcribes the coding sequence into mRNA, which isthen trans-RNA spliced (if it contains introns) and translated into theprotein encoded by the coding sequence.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell. Anexpression product can be characterized as intracellular, extracellularor secreted. The term “intracellular” means something that is inside acell. The term “extracellular” means something that is outside a cell. Asubstance is “secreted” by a cell if it appears in significant measureoutside the cell, from somewhere on or inside the cell.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell. The term “transformation” means the introduction of a“foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence toa host cell, so that the host cell will express the introduced gene orsequence to produce a desired substance, typically a protein or enzymecoded by the introduced gene or sequence. The introduced gene orsequence may also be called a “cloned” or “foreign” gene or sequence,may include regulatory or control sequences, such as start, stop,promoter, signal, secretion, or other sequences used by a cell's geneticmachinery. The gene or sequence may include nonfunctional sequences orsequences with no known function. A host cell that receives andexpresses introduced DNA or RNA has been “transformed” and is a“transformant” or a “clone.” The DNA or RNA introduced to a host cellcan come from any source, including cells of the same genus or speciesas the host cell, or cells of a different genus or species.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence. Vectors include plasmids, phages, viruses, etc.; they arediscussed in greater detail below.

Vectors typically comprise the DNA of a transmissible agent, into whichforeign DNA is inserted. A common way to insert one segment of DNA intoanother segment of DNA involves the use of enzymes called restrictionenzymes that cleave DNA at specific sites (specific groups ofnucleotides) called restriction sites. A “cassette” refers to a DNAcoding sequence or segment of DNA that codes for an expression productthat can be inserted into a vector at defined restriction sites. Thecassette restriction sites are designed to ensure insertion of thecassette in the proper reading frame. Generally, foreign DNA is insertedat one or more restriction sites of the vector DNA, and then is carriedby the vector into a host cell along with the transmissible vector DNA.A segment or sequence of DNA having inserted or added DNA, such as anexpression vector, can also be called a “DNA construct.” A common typeof vector is a “plasmid”, which generally is a self-contained moleculeof double-stranded DNA, usually of bacterial origin, that can readilyaccept additional (foreign) DNA and which can readily introduced into asuitable host cell. A plasmid vector often contains coding DNA andpromoter DNA and has one or more restriction sites suitable forinserting foreign DNA. Coding DNA is a DNA sequence that encodes aparticular amino acid sequence for a particular protein or enzyme.Promoter DNA is a DNA sequence which initiates, regulates, or otherwisemediates or controls the expression of the coding DNA. Promoter DNA andcoding DNA may be from the same gene or from different genes, and may befrom the same or different organisms. A large number of vectors,including plasmid and fungal vectors, have been described forreplication and/or expression in a variety of eukaryotic and prokaryotichosts. Non-limiting examples include pKK plasmids (Clonetech), pUCplasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREPplasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New EnglandBiolabs, Beverly, Mass.), and many appropriate host cells, using methodsdisclosed or cited herein or otherwise known to those skilled in therelevant art. Recombinant cloning vectors will often include one or morereplication systems for cloning or expression, one or more markers forselection in the host, e.g. antibiotic resistance, and one or moreexpression cassettes.

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include E. coli host cells and plasmidvectors, insect host cells and Baculovirus vectors, and mammalian hostcells and vectors. In a specific embodiment, Ozz is expressed in COS-1or C₂C₁₂ cells. Other suitable cells include CHO cells, HeLa cells, 293T(human kidney cells), mouse primary myoblasts, and NIH 3T3 cells.

The term “heterologous” refers to a combination of elements notnaturally occurring. For example, heterologous DNA refers to DNA notnaturally located in the cell, or in a chromosomal site of the cell.Preferably, the heterologous DNA includes a gene foreign to the cell. Aheterologous expression regulatory element is a such an elementoperatively associated with a different gene than the one it isoperatively associated with in nature. In the context of the presentinvention, an Ozz gene is heterologous to the vector DNA in which it isinserted for cloning or expression, and it is heterologous to a hostcell containing such a vector, in which it is expressed, e.g., a CHOcell.

The terms “mutant” and “mutation” mean any detectable change in geneticmaterial, e.g. DNA, or any process, mechanism, or result of such achange. This includes gene mutations, in which the structure (e.g. DNAsequence) of a gene is altered, any gene or DNA arising from anymutation process, and any expression product (e.g. protein or enzyme)expressed by a modified gene or DNA sequence. The term “variant” mayalso be used to indicate a modified or altered gene, DNA sequence,enzyme, cell, etc., i.e., any kind of mutant.

“Sequence-conservative variants” of a polynucleotide sequence are thosein which a change of one or more nucleotides in a given codon positionresults in no alteration in the amino acid encoded at that position.

“Function-conservative variants” are those in which a given amino acidresidue in a protein or enzyme has been changed without altering theoverall conformation and function of the polypeptide, including, but notlimited to, replacement of an amino acid with one having similarproperties (such as, for example, polarity, hydrogen bonding potential,acidic, basic, hydrophobic, aromatic, and the like). Amino acids withsimilar properties are well known in the art. For example, arginine,histidine and lysine are hydrophilic-basic amino acids and may beinterchangeable. Similarly, isoleucine, a hydrophobic amino acid, may bereplaced with leucine, methionine or valine. Such changes are expectedto have little or no effect on the apparent molecular weight orisoelectric point of the protein or polypeptide. Amino acids other thanthose indicated as conserved may differ in a protein or enzyme so thatthe percent protein or amino acid sequence similarity between any twoproteins of similar function may vary and may be, for example, from 70%to 99% as determined according to an alignment scheme such as by theCluster Method, wherein similarity is based on the MEGALIGN algorithm. A“function-conservative variant” also includes a polypeptide or enzymewhich has at least 60% amino acid identity as determined by BLAST orFASTA algorithms, preferably at least 75%, most preferably at least 85%,and even more preferably at least 90%, and which has the same orsubstantially similar properties or functions as the native or parentprotein or enzyme to which it is compared.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., Cell 50:667, 1987). Such proteins (and their encoding genes)have sequence homology, as reflected by their sequence similarity,whether in terms of percent similarity or the presence of specificresidues or motifs at conserved positions.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., supra). However, in common usageand in the instant application, the term “homologous,” when modifiedwith an adverb such as “highly,” may refer to sequence similarity andmay or may not relate to a common evolutionary origin.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 80%, and mostpreferably at least about 90 or 95%) of the nucleotides match over thedefined length of the DNA sequences, as determined by sequencecomparison algorithms, such as BLAST, FASTA, DNA Strider, etc. Anexample of such a sequence is an allelic or species variant of thespecific Ozz genes of the invention. Sequences that are substantiallyhomologous can be identified by comparing the sequences using standardsoftware available in sequence data banks, or in a Southernhybridization experiment under, for example, stringent conditions asdefined for that particular system.

Similarly, in a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when greater than80% of the amino acids are identical, or greater than about 90% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program, or any of the programs described above(BLAST, FASTA, etc)

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m)(melting temperature) of 55° C., can be used, e.g., 5×SSC, 0.1% SDS,0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS).Moderate stringency hybridization conditions correspond to a higherT_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SCC. SCC is a 0.15M NaCl, 0.015M Na-citrate.Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of T_(m) for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherT_(m)) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nt in length,equations for calculating T_(m) have been derived (see Sambrook et al.,supra, 9.50-9.51). For hybridization with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-11.8). A minimum length for a hybridizablenucleic acid is at least about 10 nt; preferably at least about 15 nt;and more preferably the length is at least about 20 nt.

In a specific embodiment, the term “standard hybridization conditions”refers to a T_(m) of 55° C., and utilizes conditions as set forth above.In a preferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 65° C. In a specific embodiment, “highstringency” refers to hybridization and/or washing conditions at 68° C.in 0.2×SSC, at 42° C. in 50% formamide, 4×SSC, or under conditions thatafford levels of hybridization equivalent to those observed under eitherof these two conditions.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 10, preferably at least 15, and more preferably atleast 20 nt, preferably no more than 100 nt, that is hybridizable to agenomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding agene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotidescan be labeled, e.g., with ³²P-nucleotides or nucleotides to which alabel, such as biotin, has been covalently conjugated. In oneembodiment, a labeled oligonucleotide can be used as a probe to detectthe presence of a nucleic acid. In another embodiment, oligonucleotides(one or both of which may be labeled) can be used as PCR primers, eitherfor cloning full length or a fragment of Ozz, or to detect the presenceof nucleic acids encoding Ozz. In a further embodiment, anoligonucleotide of the invention can form a triple helix with a Ozz DNAmolecule. Generally, oligonucleotides are prepared synthetically,preferably on a nucleic acid synthesizer. Accordingly, oligonucleotidescan be prepared with non-naturally occurring phosphoester analog bonds,such as thioester bonds, etc.

The present invention provides antisense nucleic acids (includingribozymes), which may be used to inhibit expression of Ozz of theinvention, particularly to suppress Ozz regulation of β-catenin. An“antisense nucleic acid” is a single stranded nucleic acid moleculewhich, on hybridizing under cytoplasmic conditions with complementarybases in an RNA or DNA molecule, inhibits the latter's role. If the RNAis a messenger RNA transcript, the antisense nucleic acid is acountertranscript or mRNA-interfering complementary nucleic acid. Aspresently used, “antisense” broadly includes RNA-RNA interactions,RNA-DNA interactions, ribozymes and RNase-H mediated arrest. Antisensenucleic acid molecules can be encoded by a recombinant gene forexpression in a cell (e.g., U.S. Pat. No. 5,814,500; U.S. Pat. No.5,811,234), or alternatively they can be prepared synthetically (e.g.,U.S. Pat. No. 5,780,607).

Specific non-limiting examples of synthetic oligonucleotides envisionedfor this invention include oligonucleotides that containphosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl, or cycloalkl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are those withCH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂, CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂and O—N(CH₃)—CH₂—CH₂ backbones (where phosphodiester is O—PO₂—O—CH₂).U.S. Pat. No. 5,677,437 describes heteroaromatic olignucleosidelinkages. Nitrogen linkers or groups containing nitrogen can also beused to prepare oligonucleotide mimics (U.S. Pat. No. 5,792,844 and No.5,783,682). U.S. Pat. No. 5,637,684 describes phosphoramidate andphosphorothioamidate oligomeric compounds. Also envisioned areoligonucleotides having morpholino backbone structures (U.S. Pat. No.5,034,506). In other embodiments, such as the peptide-nucleic acid (PNA)backbone, the phosphodiester backbone of the oligonucleotide may bereplaced with a polyamide backbone, the bases being bound directly orindirectly to the aza nitrogen atoms of the polyamide backbone (Nielsenet al., Science 254:1497, 1991). Other synthetic oligonucleotides maycontain substituted sugar moieties comprising one of the following atthe 2′ position: OH, SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃where n is from 1 to about 10; C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O—; S—, or N-alkyl;O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂;heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;substitued silyl; a fluorescein moiety; an RNA cleaving group; areporter group; an intercalator; a group for improving thepharmacokinetic properties of an oligonucleotide; or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Oligonucleotides may alsohave sugar mimetics such as cyclobutyls or other carbocyclics in placeof the pentofuranosyl group. Nucleotide units having nucleosides otherthan adenosine, cytidine, guanosine, thymidine and uridine, such asinosine, may be used in an oligonucleotide molecule.

Ozz Nucleic Acids

A gene encoding Ozz, whether genomic DNA or cDNA, can be isolated fromany source, particularly from a human cDNA or genomic library. Methodsfor obtaining Ozz gene are well known in the art, as described above(see, e.g., Sambrook et al., 1989, supra). The DNA may be obtained bystandard procedures known in the art from cloned DNA (e.g., a DNA“library”), and preferably is obtained from a cDNA library prepared fromtissues with high level expression of the protein (e.g., a muscle celllibrary, since these are the cells that evidence highest levels ofexpression of Ozz), by chemical synthesis, by cDNA cloning, or by thecloning of genomic DNA, or fragments thereof, purified from the desiredcell (See, for example, Sambrook et al., 1989, supra; Glover, D. M.(ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford,U.K. Vol. I, II). Clones derived from genomic DNA may contain regulatoryand intron DNA regions in addition to coding regions; clones derivedfrom cDNA will not contain intron sequences. Whatever the source, thegene should be molecularly cloned into a suitable vector for propagationof the gene. Identification of the specific DNA fragment containing thedesired Ozz gene may be accomplished in a number of ways. For example, aportion of an Ozz gene exemplified infra can be purified and labeled toprepare a labeled probe, and the generated DNA may be screened bynucleic acid hybridization to the labeled probe (Benton and Davis,Science 196:180, 1977; Grunstein and Hogness, Proc. Natl. Acad. Sci.U.S.A. 72:3961, 1975). Those DNA fragments with substantial homology tothe probe, such as an allelic variant from another individual, willhybridize. In a specific embodiment, highest stringency hybridizationconditions are used to identify a homologous Ozz gene.

Further selection can be carried out on the basis of the properties ofthe gene, e.g., if the gene encodes a protein product having theisoelectric, electrophoretic, amino acid composition, partial orcomplete amino acid sequence, antibody binding activity, or ligandbinding profile of Ozz protein as disclosed herein. Thus, the presenceof the gene may be detected by assays based on the physical, chemical,immunological, or functional properties of its expressed product.

Other DNA sequences which encode substantially the same amino acidsequence as an Ozz gene may be used in the practice of the presentinvention. These include but are not limited to allelic variants,species variants, sequence conservative variants, and functionalvariants.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys.

The genes encoding Ozz derivatives and analogs of the invention can beproduced by various methods known in the art. The manipulations whichresult in their production can occur at the gene or protein level. Forexample, the cloned Ozz gene sequence can be modified by any of numerousstrategies known in the art (Sambrook et al., 1989, supra). The sequencecan be cleaved at appropriate sites with restriction endonuclease(s),followed by further enzymatic modification if desired, isolated, andligated in vitro. In the production of the gene encoding a derivative oranalog of Ozz, care should be taken to ensure that the modified generemains within the same translational reading frame as the Ozz gene,uninterrupted by translational stop signals, in the gene region wherethe desired activity is encoded.

Additionally, the Ozz-encoding nucleic acid sequence can be mutated invitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy preexistingones, to facilitate further in vitro modification. Such modificationscan be made to introduce restriction sites and facilitate cloning theOzz gene into an expression vector. Any technique for mutagenesis knownin the art can be used, including but not limited to, in vitrosite-directed mutagenesis (Hutchinson, C., et al., J. Biol. Chem.253:6551, 1978; Zoller and Smith, DNA 3:479-488, 1984; Oliphant et al.,Gene 44:177, 1986; Hutchinson et al., Proc. Natl. Acad. Sci. U.S.A.83:710, 1986), use of TAB^(••) linkers (Pharmacia), etc. PCR techniquesare preferred for site directed mutagenesis (see Higuchi, 1989, “UsingPCR to Engineer DNA”, in PCR Technology: Principles and Applications forDNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp.61-70).

The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Examples of vectors include, but arenot limited to, E. coli, bacteriophages such as lambda derivatives, orplasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g.,pGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vectorcan, for example, be accomplished by ligating the DNA fragment into acloning vector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences.

Recombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation, etc., so thatmany copies of the gene sequence are generated. Preferably, the clonedgene is contained on a shuttle vector plasmid, which provides forexpansion in a cloning cell, e.g., E. coli, and facile purification forsubsequent insertion into an appropriate expression cell line, if suchis desired. For example, a shuttle vector, which is a vector that canreplicate in more than one type of organism, can be prepared forreplication in both E. coli and Saccharomyces cerevisiae by linkingsequences from an E. coli plasmid with sequences form the yeast 2 mplasmid.

Ozz Regulatory Nucleic Acids

A particular advantage of the present invention is the identification ofthe heart and muscle-specific promoter of Ozz. This discovery hasimportant implications in the field of gene therapy, since therapeuticvectors, as described in the sub-section entitled “Vectors”, infra, canbe modified to employ the Ozz promoter for tissue-specific expression ofa therapeutic protein. For example, expression of an angiogenic factor,such as basic fibroblast growth factor, VEGF, VEGF-2, angiopoietin,etc., can be limited to target ischemic muscle (heart or skeletalmuscle).

Ozz appears to share the PPCA proximal promoter (see FIG. 3), whichcontains three E-boxes. These E-boxes, which can function in eitherorientation, are target sites for muscle-specific transcription factorsbelonging to the Myo-D family. (It is, therefore, surprising to findthem in the PPCA promoter, since PPCA is expressed ubiquitously). Otherelements of the Ozz promoter can be identified by scanning the humangenomic region upstream of the Ozz start site, e.g., by creatingdeletion mutants and checking for expression, or with the TRANSFACalgorithm. Sequences up to about 6 kb or more upstream from the Ozzstart site can contain tissue-specific regulatory elements. Inparticular, in intron X of the human PPGB gene (the human homolog ofmurine PPCA), at 5.5 kb and 4.5 kb upstream of the Ozz start site,recognition sites for a cardiac-specific transcription factor nkx-2.5characterized in mouse (Chen, et al., J. Biol. Chem., 270:15628-33,1995) have been identified. These may function to regulatetissue-specific expression of Ozz.

The term “Ozz promoter” encompasses artificial promoters. Such promoterscan be prepared by deleting non-essentially intervening sequences fromthe upstream region of the Ozz promoter, or by joining upstreamregulatory elements from the Ozz promoter with a heterologous minimalpromoter, such as the CMV immediate early promoter.

Expression of Ozz Polypeptides

The nucleotide sequence coding for Ozz, or antigenic fragment,derivative or analog thereof, or a functionally active derivative,including a chimeric protein, thereof, can be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. Thus, a nucleic acid encoding Ozz of theinvention can be operationally associated with a promoter in anexpression vector of the invention. Both cDNA and genomic sequences canbe cloned and expressed under control of such regulatory sequences. Suchvectors can be used to express functional or functionally inactivatedOzz polypeptides.

The necessary transcriptional and translational signals can be providedon a recombinant expression vector, or they may be supplied by thenative gene encoding Ozz and/or its flanking regions.

Potential host-vector systems include but are not limited to mammaliancell systems transfected with expression plasmids or infected with virus(e.g., vaccinia virus, adenovirus, adeno-associated virus, herpes virus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

Expression of Ozz protein may be controlled by any promoter/enhancerelement known in the art, but these regulatory elements must befunctional in the host selected for expression. Promoters which may beused to control Ozz gene expression include, but are not limited to,cytomegalovirus (CMV) promoter, the SV40 early promoter region (Benoistand Chambon, 1981, Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell22:787-797, 1980), the herpes thymidine kinase promoter (Wagner et al.,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981), the regulatorysequences of the metallothionein gene (Brinster et al., Nature296:39-42, 1982); prokaryotic expression vectors such as the b-lactamasepromoter (Villa-Komaroff, et al., Proc. Natl. Acad. Sci. U.S.A.75:3727-3731, 1978), or the tac promoter (DeBoer, et al., Proc. Natl.Acad. Sci. U.S.A. 80:21-25, 1983); see also “Useful proteins fromrecombinant bacteria” in Scientific American, 242:74-94, 1980; promoterelements from yeast or other fungi such as the Gal 4 promoter, the ADC(alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter,alkaline phosphatase promoter; and transcriptional control regions thatexhibit hematopoietic tissue specificity, in particular: beta-globingene control region which is active in myeloid cells (Mogram et al.,Nature 315:338-340, 1985; Kollias et al., Cell 46:89-94, 1986),hematopoietic stem cell differentiation factor promoters, erythropoietinreceptor promoter (Maouche et al., Blood, 15:2557, 1991), etc.

Soluble forms of the protein can be obtained by collecting culturefluid, or solubilizing inclusion bodies, e.g., by treatment withdetergent, and if desired sonication or other mechanical processes, asdescribed above. The solubilized or soluble protein can be isolatedusing various techniques, such as polyacrylamide gel electrophoresis(PAGE), isoelectric focusing, 2-dimensional gel electrophoresis,chromatography (e.g., ion exchange, affinity, immunoaffinity, and sizingcolumn chromatography), centrifugation, differential solubility,immunoprecipitation, or by any other standard technique for thepurification of proteins.

Vectors

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol El, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al., Gene 67:31-40,1988), pMB9 and their derivatives, plasmids such as RP4; phage DNAS,e.g., the numerous derivatives of phage 1, e.g., NM989, and other phageDNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmidssuch as the 2 m plasmid or derivatives thereof; vectors useful ineukaryotic cells, such as vectors useful in insect or mammalian cells;vectors derived from combinations of plasmids and phage DNAs, such asplasmids that have been modified to employ phage DNA or other expressioncontrol sequences; and the like.

Preferred vectors are viral vectors, such as lentiviruses, retroviruses,herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus,baculovirus, and other recombinant viruses with desirable cellulartropism. Thus, a gene encoding a functional or mutant Ozz protein orpolypeptide domain fragment thereof can be introduced in vivo, ex vivo,or in vitro using a viral vector or through direct introduction of DNA.Expression in targeted tissues can be effected by targeting thetransgenic vector to specific cells, such as with a viral vector or areceptor ligand, or by using a tissue-specific promoter, or both.Targeted gene delivery is described in International Patent PublicationWO 95/28494, published October 1995.

Viral vectors commonly used for in vivo or ex vivo targeting and therapyprocedures are DNA-based vectors and retroviral vectors. Methods forconstructing and using viral vectors are known in the art (see, e.g.,Miller and Rosman, BioTechniques, 7:980-990, 1992). Preferably, theviral vectors are replication defective, that is, they are unable toreplicate autonomously in the target cell. In general, the genome of thereplication defective viral vectors which are used within the scope ofthe present invention lack at least one region which is necessary forthe replication of the virus in the infected cell. These regions caneither be eliminated (in whole or in part), be rendered non-functionalby any technique known to a person skilled in the art. These techniquesinclude the total removal, substitution (by other sequences, inparticular by the inserted nucleic acid), partial deletion or additionof one or more bases to an essential (for replication) region. Suchtechniques may be performed in vitro (on the isolated DNA) or in situ,using the techniques of genetic manipulation or by treatment withmutagenic agents. Preferably, the replication defective virus retainsthe sequences of its genome which are necessary for encapsidating theviral particles.

DNA viral vectors include an attenuated or defective DNA virus, such asbut not limited to herpes simplex virus (HSV), papillomavirus, EpsteinBarr virus (EBV), adenovirus, adeno-associated virus (AAV), and thelike. Defective viruses, which entirely or almost entirely lack viralgenes, are preferred. Defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt etal., Molec. Cell. Neurosci. 2:320-330, 1991), defective herpes virusvector lacking a glyco-protein L gene (Patent Publication RD 371005 A),or other defective herpes virus vectors (International PatentPublication No. WO 94/21807, published Sep. 29, 1994; InternationalPatent Publication No. WO 92/05263, published Apr. 2, 1994); anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al. (J. Clin. Invest. 90:626-630, 1992; seealso La Salle et al., Science 259:988-990, 1993); and a defectiveadeno-associated virus vector (Samulski et al., J. Virol. 61:3096-3101,1987; Samulski et al., J. Virol. 63:3822-3828, 1989; Lebkowski et al.,Mol. Cell. Biol. 8:3988-3996, 1988).

Various companies produce viral vectors commercially, including but byno means limited to Avigen, Inc. (Alameda, Calif.; AAV vectors), CellGenesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, andlentiviral vectors), Clontech (retroviral and baculoviral vectors),Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviralvectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors).

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector, e.g.,adenovirus vector, to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-g (IFN-g), or anti-CD4 antibody, canbe administered to block humoral or cellular immune responses to theviral vectors (see, e.g., Wilson, Nature Medicine, 1995). In thatregard, it is advantageous to employ a viral vector that is engineeredto express a minimal number of antigens.

Ozz Binding Partners

The present invention further permits identification of physiologicalbinding partners of Ozz. For example, as shown below, the inventionexemplifies reagents to investigate the interaction between Ozz andβ-catenin. Similar experiments can be done with myosin and the lessknown c-Nap and Alix, in order to understand their possible role in theOzz pathway.

One method for evaluating and identifying Ozz binding partners is theyeast two-hybrid screen. Preferably, the yeast two-hybrid screen wouldbe performed using a muscle cell library with yeast that are transformedwith recombinant Ozz, e.g., as shown in the Example, infra.Alternatively, Ozz can be used as a capture or affinity purificationreagent. Again, the preferred source material for such preparations ismuscle cells. In another alternative, labeled Ozz can be used as a probefor binding, e.g., immunoprecipitation or Western analysis.

Generally, binding interactions between Ozz and any of its bindingpartners will be strongest under conditions approximating those found inthe cytoplasm, i.e., physiological conditions of ionic strength, pH andtemperature. Perturbation of these conditions will tend to disrupt thestability of a binding interaction.

Antibodies to Ozz

Antibodies to Ozz are useful, inter alia, for diagnostics andintracellular regulation of Ozz activity, as set forth below. Accordingto the invention, Ozz polypeptides produced recombinantly or by chemicalsynthesis, and fragments or other derivatives or analogs thereof,including fusion proteins, may be used as an immunogen to generateantibodies that recognize the Ozz polypeptide. Such antibodies includebut are not limited to polyclonal, monoclonal, chimeric, single chain,Fab fragments, and an Fab expression library. Such an antibody isspecific for human Ozz; it may recognize a mutant form of Ozz, orwild-type Ozz.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to Ozz polypeptide or derivative or analogthereof. For the production of antibody, various host animals can beimmunized by injection with the Ozz polypeptide, or a derivative (e.g.,fragment or fusion protein) thereof, including but not limited torabbits, mice, rats, sheep, goats, etc. In one embodiment, the Ozzpolypeptide or fragment thereof can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the Ozzpolypeptide, or fragment, analog, or derivative thereof, any techniquethat provides for the production of antibody molecules by continuouscell lines in culture may be used. These include but are not limited tothe hybridoma technique originally developed by Kohler and Milstein(Nature 256:495-497, 1975), as well as the trioma technique, the humanB-cell hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983;Cote et al., Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030, 1983), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96, 1985). In an additional embodiment of the invention, monoclonalantibodies can be produced in germ-free animals (International PatentPublication No. WO 89/12690, published 28 Dec. 1989). In fact, accordingto the invention, techniques developed for the production of “chimericantibodies” (Morrison et l., J. Bacteriol. 159:870, 1984); Neuberger etal., Nature 312:604-608, 1984; Takeda et al., Nature 314:452-454, 1985)by splicing the genes from a mouse antibody molecule specific for an Ozzpolypeptide together with genes from a human antibody molecule ofappropriate biological activity can be used; such antibodies are withinthe scope of this invention. Such human or humanized chimeric antibodiesare preferred for use in therapy of human diseases or disorders(described infra), since the human or humanized antibodies are much lesslikely than xenogenic antibodies to induce an immune response, inparticular an allergic response, themselves.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. Nos. 5,476,786 and 5,132,405 toHuston; U.S. Pat. No. 4,946,778) can be adapted to produce Ozzpolypeptide-specific single chain antibodies. An additional embodimentof the invention utilizes the techniques described for the constructionof Fab expression libraries (Huse et al., Science 246:1275-1281, 1989)to allow rapid and easy identification of monoclonal Fab fragments withthe desired specificity for an Ozz polypeptide, or its derivatives, oranalogs.

In the production and use of antibodies, screening for or testing withthe desired antibody can be accomplished by techniques known in the art,e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),“sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention. For example, to select antibodies whichrecognize a specific epitope of an Ozz polypeptide, one may assaygenerated hybridomas for a product which binds to an Ozz polypeptidefragment containing such epitope. For selection of an antibody specificto an Ozz polypeptide from a particular species of animal, one canselect on the basis of positive binding with Ozz polypeptide expressedby or isolated from cells of that species of animal.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the Ozz polypeptide, e.g.,for Western blotting, imaging Ozz polypeptide in situ, measuring levelsthereof in appropriate physiological samples, etc. using any of thedetection techniques mentioned above or known in the art. Suchantibodies can also be used in assays for ligand binding, e.g., asdescribed in U.S. Pat. No. 5,679,582. Antibody binding generally occursmost readily under physiological conditions, e.g., pH of between about 7and 8, and physiological ionic strength. The presence of a carrierprotein in the buffer solutions stabilizes the assays. While there issome tolerance of perturbation of optimal conditions, e.g., increasingor decreasing ionic strength, temperature, or pH, or adding detergentsor chaotropic salts, such perturbations will decrease binding stability.

In a specific embodiment, antibodies that agonize or antagonize theactivity of Ozz polypeptide can be generated. In particular,intracellular single chain Fv antibodies can be used to regulate(inhibit) Ozz. Such antibodies can be tested using the assays describedinfra for identifying ligands.

Screening and Chemistry

According to the present invention, nucleotide sequences derived fromthe gene encoding Ozz, and peptide sequences derived from Ozz, areuseful targets to identify drugs that are effective in treatingmyogenesis disorders. Drug targets include without limitation (i)isolated nucleic acids derived from the gene encoding Ozz and (ii)isolated peptides and polypeptides derived from Ozz polypeptides.

In particular, identification and isolation of Ozz provides fordevelopment of screening assays, particularly for high throughputscreening of molecules that up- or down-regulate the activity of Ozz,e.g., by permitting expression of Ozz in quantities greater than can beisolated from natural sources, or in indicator cells that are speciallyengineered to indicate the activity of Ozz expressed after transfectionor transformation of the cells. Accordingly, the present inventioncontemplates methods for identifying specific ligands of Ozz usingvarious screening assays known in the art.

Any screening technique known in the art can be used to screen for Ozzagonists or antagonists. The present invention contemplates screens forsmall molecule ligands or ligand analogs and mimics, as well as screensfor natural ligands that bind to and agonize or antagonize Ozz in vivo.Such agonists or antagonists may, for example, interfere in thephosphorylation or dephosphorylation of Ozz, with resulting effects onOzz function. For example, natural products libraries can be screenedusing assays of the invention for molecules that agonize or antagonizeOzz activity.

Knowledge of the primary sequence of Ozz, and the similarity of thatsequence with proteins of known function, can provide an initial clue asthe inhibitors or antagonists of the protein. Identification andscreening of antagonists is further facilitated by determiningstructural features of the protein, e.g., using X-ray crystallography,neutron diffraction, nuclear magnetic resonance spectrometry, and othertechniques for structure determination. These techniques provide for therational design or identification of agonists and antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” (Scott and Smith, Science249:386-390, 1990; Cwirla, et al., Proc. Natl. Acad. Sci., 87:6378-6382,1990; Devlin et al., Science, 49:404-406, 1990), very large librariescan be constructed (10⁶-10⁸ chemical entities). A second approach usesprimarily chemical methods, of which the Geysen method (Geysen et al.,Molecular Immunology 23:709-715, 1986; Geysen et al. J. ImmunologicMethod 102:259-274, 1987; and the method of Fodor et al. (Science251:767-773, 1991) are examples. Furka et al. (14th InternationalCongress of Biochemistry, Volume #5, Abstract FR:013, 1988; Furka, Int.J. Peptide Protein Res. 37:487-493, 1991), Houghton (U.S. Pat. No.4,631,211, issued December 1986) and Rutter et al. (U.S. Pat. No.5,010,175, issued Apr. 23, 1991) describe methods to produce a mixtureof peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries (Needels et al., Proc. Natl.Acad. Sci. USA 90:10700-4, 1993; Ohlmeyer et al., Proc. Natl. Acad. Sci.USA 90:10922-10926, 1993; Lam et al., International Patent PublicationNo. WO 92/00252; Kocis et al., International Patent Publication No. WO9428028) and the like can be used to screen for Ozz ligands according tothe present invention.

Test compounds are screened from large libraries of synthetic or naturalcompounds. Numerous means are currently used for random and directedsynthesis of saccharide, peptide, and nucleic acid based compounds.Synthetic compound libraries are commercially available from MaybridgeChemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.),Brandon Associates (Merrimack, N.H.), and Microsource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant and animal extracts are available from e.g. PanLaboratories (Bothell, Wash.) or MycoSearch (NC), or are readilyproducible. Additionally, natural and synthetically produced librariesand compounds are readily modified through conventional chemical,physical, and biochemical means (Blondelle et al., Tib Tech, 14:60,1996).

In Vivo Screening Methods

Intact cells or whole animals expressing a gene encoding Ozz can be usedin screening methods to identify candidate drugs.

In one series of embodiments, a permanent cell line is established.Alternatively, cells (including without limitation mammalian, insect,yeast, or bacterial cells) are transiently programmed to express an Ozzgene by introduction of appropriate DNA or mRNA. Identification ofcandidate compounds can be achieved using any suitable assay, includingwithout limitation (i) assays that measure selective binding of testcompounds to Ozz (ii) assays that measure the ability of a test compoundto modify (i.e., inhibit or enhance) a measurable activity or functionof Ozz and (iii) assays that measure the ability of a compound to modify(i.e., inhibit or enhance) the transcriptional activity of sequencesderived from the promoter (i.e., regulatory) regions the Ozz gene.

Ozz knockout mammals can be prepared for evaluating the molecularpathology of this defect in greater detail than is possible with humansubjects. Such animals also provide excellent models for screening drugcandidates. A “knockout mammal” is an mammal (e.g., mouse) that containswithin its genome a specific gene that has been inactivated by themethod of gene targeting (see, e.g., U.S. Pat. No. 5,777,195 and No.5,616,491). A knockout mammal includes both a heterozygote knockout(i.e., one defective allele and one wild-type allele) and a homozygousmutant (i.e., two defective alleles; however, in this case aheterologous construct for expression of an Ozz, such as a human Ozz,would be inserted to permit the knockout mammal to live). Preparation ofa knockout mammal requires first introducing a nucleic acid constructthat will be used to suppress expression of a particular gene into anundifferentiated cell type termed an embryonic stem cell. This cell isthen injected into a mammalian embryo. A mammalian embryo with anintegrated cell is then implanted into a foster mother for the durationof gestation. Zhou, et al. (Genes and Development, 9:2623-34, 1995)describes PPCA knock-out mice.

The term “knockout” refers to partial or complete suppression of theexpression of at least a portion of a protein encoded by an endogenousDNA sequence in a cell. The term “knockout construct” refers to anucleic acid sequence that is designed to decrease or suppressexpression of a protein encoded by endogenous DNA sequences in a cell.The nucleic acid sequence used as the knockout construct is typicallycomprised of (1) DNA from some portion of the gene (exon sequence,intron sequence, and/or promoter sequence) to be suppressed and (2) amarker sequence used to detect the presence of the knockout construct inthe cell. The knockout construct is inserted into a cell, and integrateswith the genomic DNA of the cell in such a position so as to prevent orinterrupt transcription of the native DNA sequence. Such insertionusually occurs by homologous recombination (i.e., regions of theknockout construct that are homologous to endogenous DNA sequenceshybridize to each other when the knockout construct is inserted into thecell and recombine so that the knockout construct is incorporated intothe corresponding position of the endogenous DNA). The knockoutconstruct nucleic acid sequence may comprise 1) a full or partialsequence of one or more exons and/or introns of the gene to besuppressed, 2) a full or partial promoter sequence of the gene to besuppressed, or 3) combinations thereof. Typically, the knockoutconstruct is inserted into an embryonic stem cell (ES cell) and isintegrated into the ES cell genomic DNA, usually by the process ofhomologous recombination. This ES cell is then injected into, andintegrates with, the developing embryo.

The phrases “disruption of the gene” and “gene disruption” refer toinsertion of a nucleic acid sequence into one region of the native DNAsequence (usually one or more exons) and/or the promoter region of agene so as to decrease or prevent expression of that gene in the cell ascompared to the wild-type or naturally occurring sequence of the gene.By way of example, a nucleic acid construct can be prepared containing aDNA sequence encoding an antibiotic resistance gene which is insertedinto the DNA sequence that is complementary to the DNA sequence(promoter and/or coding region) to be disrupted. When this nucleic acidconstruct is then transfected into a cell, the construct will integrateinto the genomic DNA. Thus, many progeny of the cell will no longerexpress the gene at least in some cells, or will express it at adecreased level, as the DNA is now disrupted by the antibioticresistance gene.

Generally, the DNA will be at least about 1 kilobase (kb) in length andpreferably 3-4 kb in length, thereby providing sufficient complementarysequence for recombination when the knockout construct is introducedinto the genomic DNA of the ES cell (discussed below).

Included within the scope of this invention is a mammal in which two ormore genes have been knocked out. Such mammals can be generated byrepeating the procedures set forth herein for generating each knockoutconstruct, or by breeding to mammals, each with a single gene knockedout, to each other, and screening for those with the double knockoutgenotype.

Regulated knockout animals can be prepared using various systems, suchas the tet-repressor system (see U.S. Pat. No. 5,654,168) or the Cre-Loxsystem (see U.S. Pat. No. 4,959,317 and No. 5,801,030).

In another series of embodiments, transgenic animals are created inwhich (i) a human Ozz is stably inserted into the genome of thetransgenic animal; and/or (ii) the endogenous Ozz genes are inactivatedand replaced with human Ozz genes. See, e.g., Coffman, Semin. Nephrol.17:404, 1997; Esther et al., Lab. Invest. 74:953, 1996; Murakami et al.,Blood Press. Suppl. 2:36, 1996. Such animals can be treated withcandidate compounds and monitored for muscle weakness, heart defects, orother indicia of muscle dysfunction.

High-Throughput Screen

Agents according to the invention may be identified by screening inhigh-throughput assays, including without limitation cell-based orcell-free assays. It will be appreciated by those skilled in the artthat different types of assays can be used to detect different types ofagents. Several methods of automated assays have been developed inrecent years so as to permit screening of tens of thousands of compoundsin a short period of time. Such high-throughput screening methods areparticularly preferred. The use of high-throughput screening assays totest for agents is greatly facilitated by the availability of largeamounts of purified polypeptides, as provided by the invention.

Methods of Diagnosis

According to the present invention, genetic variants of Ozz can bedetected to diagnose a muscle degenerative disease. The various methodsfor detecting such variants are described herein. Where such variantsimpact Ozz function, either as a result of a mutated amino acid sequenceor because the mutation results in expression of a truncated protein, orno expression at all, they are expected to result in disregulation ofmuscle development or function, including, possibly, muscle degenerationor alternatively hyperproliferation (e.g., a muscle sarcoma). In anotherembodiment, the presence of Ozz in blood or a blood fraction (serum,plasma) indicates muscle tissue damage, e.g., ischemia associated witheither unstable angina, myocardial infarction, or both (see U.S. Pat.Nos. 5,747,274 and 5,744,358).

According to the present invention, altered Ozz protein levels andlocalization can be detected to diagnose diseases associated withaltered Ozz protein expression and localization. The methods fordetecting such altered protein levels and protein are described herein.When altered protein levels or protein localization are detected, theyare expected to be associated with disease states that occur withaltered Ozz expression. In one specific embodiment, the altered Ozzprotein expression and localization is associated withgalactosialidosis. In another embodiment, the altered protein levels orlocalization are evaluated with muscle cells that are from the atrium ofthe heart.

A “sample” as used herein refers to a biological sample, such as, forexample, tissue (or cells) or fluid isolated from an individual or fromin vitro cell culture constituents, as well as samples obtained from theenvironment or laboratory procedures. Non-limiting examples of cellsources available in clinical practice include without limitation musclebiopsies.

Nucleic Acid Assays

The DNA may be obtained from any cell source. DNA is extracted from thecell source or body fluid using any of the numerous methods that arestandard in the art. It will be understood that the particular methodused to extract DNA will depend on the nature of the source. Generally,the minimum amount of DNA to be extracted for use in the presentinvention is about 25 pg (corresponding to about 5 cell equivalents of agenome size of 4×10⁹ base pairs). Sequencing methods are described indetail, supra.

In another alternate embodiment, RNA is isolated from biopsy tissueusing standard methods well known to those of ordinary skill in the artsuch as guanidium thiocyanate-phenol-chloroform extraction (Chomocyznskiet al., Anal. Biochem., 162:156, 1987). The isolated RNA is thensubjected to coupled reverse transcription and amplification bypolymerase chain reaction (RT-PCR), using specific oligonucleotideprimers that are specific for a selected site. Conditions for primerannealing are chosen to ensure specific reverse transcription andamplification; thus, the appearance of an amplification product isdiagnostic of the presence of a particular genetic variation. In anotherembodiment, RNA is reverse-transcribed and amplified, after which theamplified sequences are identified by, e.g., direct sequencing. In stillanother embodiment, cDNA obtained from the RNA can be cloned andsequenced to identify a mutation.

Protein Assays

In an alternate embodiment, biopsy tissue is obtained from a subject.Antibodies that are capable of specifically binding to Ozz are thencontacted with samples of the tissue to determine the presence orabsence of a Ozz polypeptide specified by the antibody. The antibodiesmay be polyclonal or monoclonal, preferably monoclonal. Measurement ofspecific antibody binding to cells may be accomplished by any knownmethod, e.g., quantitative flow cytometry, enzyme-linked orfluorescence-linked immunoassay, Western analysis, etc.

Immunoassay technology, e.g., as described in U.S. Pat. Nos. 5,747,274and 5,744,358, and particularly solid phase “chromatographic” formatimmunoassays, are preferred for detecting proteins in blood or bloodfractions.

EXAMPLES

The present invention will be better understood by reference to thefollowing Examples, which are provided by way of exemplification and notby way of limitation.

Example 1 Discovery of Ozz

Through the analysis of the 5′ regions of the murine PPCA gene, weidentified a new transcriptional unit that overlaps with exon Ia of themurine PPCA gene and is transcribed from the opposite strand. Theresulting 1.0 kb mRNA is preferentially expressed in the heart andskeletal muscle, and encodes a protein of 285 amino acids that sharessignificant homology with the Drosophila neuralized gene. We have termedthis new protein Ozz.

Materials and Methods

Cloning. A murine heart poly A⁺ cDNA library was obtained from Clontechand screened by hybridization at high stringency (last washing step was65° C. with 0.1% of SSC) according to the manufacturer's protocol withthe PPCA exon Ia as a probe. Double positives were rescreened with thesame probe and a PPCA cDNA fragment. Phages that were only positive forthe exon Ia probe were further characterized. The isolated clones weresubcloned into pBluescript II KS (Stratagene) using standard cloningtechniques and sequenced with the Amersham thermocycler kit. A SalIfragment, containing the full length Ozz cDNA was subcloned into theeukaryotic expression vector pSCTOP, with or without the hemagglutinintag. For the generation of GST-Ozz fusion proteins we used the pGEX4T-2plasmid from Pharmacia using standard restriction and ligationprocedures.

Northern blot analysis. Northern blots containing mRNAs from mousetissues or different embryonal stages were purchased from Clontech andhybridized using the manufacturer's EXPRESSHYB mix and protocol.

Cell culture. COS-1 and C₂C₁₂ cells were cultured under standardconditions, using Dulbecco's Modified Eagle's Medium (DMEM) supplementedwith antibiotics and 5-10% Fetal Bovine Serum (FBS). Primary mousemyoblasts were isolated from 3-day-old mice and were grown in mediumconsisting of Ham's F-10 nutrient mixture, 20% FBS and 2.5 ng/ml basicFibroblast Growth Factor (bFGF). Primary myoblast and C₂C₁₂ were inducedto form myotubes by replacing their growth medium with thedifferentiation medium (DMEM plus 2% horse serum for the myoblasts andDMEM plus Insulin Transferrin Selenite (ITS) for the C₂C₁₂ cells).

Antibody generation and purification. The full-length Ozz cDNA wassubcloned into an inducible bacterial expression vector containing theglutathione S-transferase (GST) gene to generate antibodies in order tofurther characterize this novel gene product. The overexpressed GST-Ozzfusion protein was isolated using preparative SDS-PAGE and the purifiedelectro-eluted protein was subcutaneously injected into rabbits to boostthe production of anti-Ozz antibodies (made at Rockland Laboratories,Gilbertsville, Pa.). Rabbit serum was tested using protein extractsobtained from COS-1 cells transfected with the full-length Ozz cDNA.

The anti-Ozz antibodies were purified using bacterially overexpressedGST-Ozz protein. The fusion protein was purified by absorption withglutathione-agarose beads and then immobilized on AminoLink gel(PIERCE). The antiserum was passed through the column containing the Ozzprotein and the bound antibodies were eluted.

Western blot analysis and immunoprecipitation. C₂C₁₂ and COS-1 cellswere transfected with Qiagen's Superfect according to the manufacturer'sprocedures.

Protein extracts from mouse heart tissue were made by homogenizing thesamples in four volumes of NP-40 buffer (50 mM Tris-HCl pH 7.5, 250 mMNaCl, 1 mM EDTA, 1% NP-40 and proteinase inhibitor). The proteinconcentration was measured with bicinchoninic acid (BCA) according tothe manufacturer's protocol (Pierce Chemical Co.). Protein extracts from10 adult mice hearts were subjected to gel filtration on a Sephacryl-300HR and the FPLC fractions were collected. Both Ozz and β-cateninco-eluted around fraction 12 (about 500 kDa) as shown by probing Westernblots of the eluted fractions with the corresponding antibodies (datanot shown). For Western blot analysis, protein samples were resolved on12.5% SDS-polyacrylamide gels, transferred to Immobilon PVDF membranes(Millipore) and probed with anti-β catenin and anti-Ozz antibodies.

For the immunoprecipitation studies, transfected COS-1 and C₂C₁₂ cellswere seeded in 85 mm Petri dishes and metabolically labeled for 16 hourswith 350 μCi ³H-[4,5]-Leucine (Amersham). Radiolabeled proteins or FPLCfractions were immunoprecipitated with the indicated antibodies, asdescribed previously (Proia, et al., J. Biol. Chem., 259:3350-54, 1984).

Yeast Two-Hybrid Screening. Standard PCR reactions were used to subclonethe coding region of Ozz (845 bp) in frame with the GAL4 DNA-bindingdomain in the “bait” vector (pPC97). The yeast strain used in thetwo-hybrid screening was MAV103. Yeast transformations were made usingthe lithium acetate procedure (Ausubel, et al., Current Protocols inMolecular Biology, New York:John Wiley & Sons, Vol. 2, Ch. 13, 1994).MAV103 harboring the GAL4 DNA-binding domain-Ozz plasmid was furthertransformed with 1 μg of an oligo (dT)-primed 14 days embryo (head and ⅓top of spine) mouse cDNA library cloned into the GAL4-activating domainvector (pPC86).

Double transformants were grown on Sc-Leu-Trp plates and subsequentlyreplica-plated onto Sc-Leu-Trp-His containing 25 or 50 mM3-Amino-1,2,4-Triazole (3-AT). After selection, transformants werere-screened on Sc-Leu-Trp-His+3AT (50 and 100 mM), on Sc-Leu-Trp-Ura andtheir ability to produce β-galactosidase was screened by the filterX-Gal assay (Chevray and Nathans, Proc. Natl. Acad. Sci. USA,89:5789-93, 1992).

Results

Cloning a novel gene. Hybridization of a mouse multi-tissue Northernblot with PPCA exon Ia as a probe revealed the presence of a non-PPCAtranscript of 1.0 kb, expressed primarily in mouse heart. Thetranscriptional orientation of the gene is opposite to that of the PPCAgene and completely overlaps with the exon Ia of PPCA (FIG. 1). Itscorresponding cDNA, isolated from a mouse heart library, included ashort 5′UTR of 20 nt, an ATG start codon in a favorable Kozak context(Kozak, Mamm. Genome, 7:563-572, 1996), an open reading frame of 855 ntand a 3′UTR of 144 nt followed by a poly A tail of 17 adenosines (FIG.2A). The cDNA is encoded by two exons, of 762 and 257 nt respectively,separated by an intron of at least 1.7 kb (FIG. 1).

The human genomic sequence of Ozz was identified by sequence comparisonof the human locus corresponding to the murine PPCA locus. Thecorresponding human (syntenic) locus is termed PPGB, and is found onchromosome 20. The sequence of PPGB is found in NCBI database underaccession number a1008726.

Specifically, sequence comparison of PPGB with the first exon of thecloned mouse Ozz DNA sequence permitted identification of the humanhomolog. The cDNA sequences of human (SEQ ID NO:3) and murine Ozz (SEQID NO:1) are highly homologous, with 85% sequence identity by BLASTNanalysis (FIG. 3).

The cDNA (FIG. 2A, mouse and 2B, human) encodes a novel protein termedOzz containing 285 amino acids with a calculated molecular weight of31.5 kDa and a predicted isoelectric point at pH 8.15. The deduced aminoacid sequence of human Ozz (SEQ ID NO:4) shows even greater homology tomurine Ozz (SEQ ID NO:2). When analyzed by BLASTP analysis, these twoproteins share 90% sequence identity and 92% sequence similarity(conservative amino acid substitutions). The comparison of these twosequences is shown in FIG. 4. Computer analysis of the protein sequenceidentified putative phosphorylation sites for casein kinase II, proteinkinase C and tyrosine kinase, and three potential myristoylation sites(FIG. 4).

The C-terminus of Ozz is homologous to members of the CIS/SOCS/JAB/SSIfamily of cytokine inducible suppressor of cytokine signalling (SOCS)proteins. This homology is restricted to a region known as the SOCS box(Starr, et al. Nature, 387:917-921, 1997; Hilton et al. Proc. Natl. Sci.USA, 95:114-119, 1998). In the Ozz Sequence, the SOCS box has the aminoacid sequence: PSLQTLCRLVIQRSMVHRLAIDGLHLPKELKDFCKYE (SEQ ID NO:23). Inthe described Ozz amino acid sequence, the bold amino acids areconserved between the Ozz protein and the SOCS box consensus sequence,the underlined amino acids represents positions that can accommodate anyhydrophobic amino acid substitution, and the double underlined aminoacids represents positions that can accommodate any amino acidsubstitution. The other residues vary within a limited number ofchoices.

Recent findings have suggested that the SOCS-box containing proteins areimplicated in binding and in turn coupling specific substrate proteinsto the ubiquitination/proteosomal pathway (Kamura, et al. Genes andDevelop., 12:3872-3881, 1998; Zhang, et al., Proc. Natl. Acad. Sci. USA,96:12436-12441, 1999), therefore controlling their intracellularturnover. This model is supported by the evidence of a directinteraction between SOCS proteins and component of the E3 ubiquitinligase complexes. In particular, SOCS 3 was shown to bind to the ElonginB/C complex via a short consensus sequence present at the N-terminus ofthe SOCS box domain and called the BC box which has the consensussequence of SLxxxCxxxI (SEQ ID NO:24). In the consensus sequence, thisindicates that the serine position can be either serine or threonine,followed by a leucine or methionine, followed by any three amino acids,followed by cystine or serine, followed by any three amino acids,followed by valine, isoleucine, or leucine.

Data base searches for homologous proteins revealed a striking homologywith the neuralized proteins of Drosophila melanogaster and D. virilis(Boulianne, et al., EMBO J., 2:2586-89, 1991; Price, et al., EMBO J.,12:2411-8, 1993; Zhou and Boulianne, Genome, 37:840-7, 1994), as well aswith the human and C. elegans homologs (Nakamura, et al., Oncogene,16:1009-19, 1998). As seen in FIG. 5, the N-terminal 100 amino acids ofthe Ozz protein share 34-44% identity with the duplicated NHRs ofneuralized proteins (Neuralized Homology Repeat, see FIGS. 5 and 6, andNakamura, supra). Furthermore, a small stretch of about 30 amino acids,located at the C-terminus of Ozz bears homology to two regions ofneuralized proteins, downstream of each of the two NHR domains. FIG. 6shows the sequence alignment between the Ozz protein and D. virilisneuralized protein relative to the four homologous domains describedearlier.

Northern blot hybridization with Ozz. The expression pattern of Ozz indifferent adult mouse tissues was examined by Northern blothybridization using the full-length cDNA as probe. Expression of theexpected 1.0 kb Ozz transcript was confined to heart and skeletalmuscle. The same expression pattern was observed in human RNApreparations from different tissues, where the 1 kb Ozz transcript wasdetected exclusively in heart and skeletal muscle, but not in thepancreas, kidney, liver, lung, placenta, or brain (FIG. 7).

It is indicative that the constitutive promoter of PPCA, which likelycontrols the transcription of the Ozz gene, contains three conservedE-boxes that are target sites for muscle-specific transcription factorsbelonging to the Myo-D family that control muscle development anddifferentiation (FIG. 3). In keeping with this observation, we foundthat Ozz mRNA starts to be detected in the embryo between day E12 andE15.

Protein expression. To gain insights into the precise timing and patternof expression of the Ozz protein, as well as its physiological role, weraised mono specific polyclonal antibodies against a bacteriallyexpressed full length protein fused to glutathion-S-transferase(GST-OZZ). These antibodies have been particularly useful forimmunocytochemistry and immunoprecipitation studies.

Anti-Ozz antibodies were first tested for their ability to recognize theOzz protein overexpressed in two different cell lines: COS-1 and C₂C₁₂(the latter is a transformed mouse myoblast cell line). The antibodiesimmunoprecipitated a 29-30 kDa Ozz protein from both cell linestransfected with an Ozz cDNA construct (FIG. 8). The nature of thehigher molecular weight band recognized by the Ozz antibodies in COS-1cells is at the moment unclear.

Immunostaining of paraffin embedded sections of different mouse tissuesclearly demonstrated a high level of expression of the protein incardiac and skeletal muscle. These results confirmed the tissuedistribution of the Ozz mRNA. In similar immunohystochemistryexperiments, we could detect the Ozz protein in the mouse embryo asearly as embryonal day 10 (E10). At this stage the protein isspecifically expressed in the myotomes and in the developing heart. AtE14.5, the protein is still detected in the developing heart and in thedifferentiating myofibers. In differentiating muscle fibers thestrongest Ozz immunostining was seen in a region juxtaposed to themyotendinous region, as demonstrated by the co-staining with nestin amarker specific for the myotendinous junctions.

Effects on Ozz during in vitro differentiation. To elucidate the role ofOzz in muscle development and differentiation, we set up an inducible invitro system, using both the transformed C₂C₁₂ muscle cell line as wellas primary mouse myoblasts isolated from 3-day-old pups. Both cell typescan be maintained in culture in an undifferentiated state and can beinduced to differentiate by serum deprivation. The level of expressionof Ozz was monitored during the differentiation process. In bothinstances the amount of Ozz protein sharply increased upon celldifferentiation. This increase was particularly evident for the primarymyoblasts. In the same cultures, we also followed the expression ofmarkers specific for muscle differentiation (i.e. myogenin and myosin).The increase of Ozz expression coincided with the appearance of myosinand decrease of myogenin. Taken together these results point to the Ozzprotein as a potential marker for terminal muscle differentiation,positioning it together with myosin downstream of the MyoD pathway.

Identification of Interacting Proteins. In order to identify possiblepartners of Ozz, that could contribute to or complement its function, weperformed a yeast two-hybrid screening using the Ozz cDNA as a bait anda cDNA library from mouse embryonal stage E14.5. Four potentiallyinteracting proteins were identified: β-catenin, myosin, c-Nap I andAlix. In line with this initial observation we were able to detect Ozz,β-catenin and myosin in the same, high molecular weight fraction (407kDa), after gel-filtration of crude extracts of adult mouse heartfractionated on an FPLC column. The interaction with β-catenin wasconfirmed by co-precipitation of Ozz with anti-β-catenin antibody fromthe 407-kDa FPLC fraction. The two proteins could also beco-precipitated from cell lysates of COS-1 cells transfected with bothcDNA constructs.

The presence of a consensus BC-box at the N-terminus of the SOCS-box ofOzz (see FIG. 4) implied a possible interaction of Ozz with the ElonginsB/C complex. To test this hypothesis, we co-expressed in 293T cellseither a full-length Ozz cDNA or a mutant cDNA lacking the completeSOCS-box (Ozz) together with a Myc-tagged Elongin B and a HPC4-taggedElongin C constructs. The ability of the expressed proteins to interactwas assessed in co-immunoprecipitation assays. Ozz was readilyco-precipitated with Elongin B, using a monoclonal anti-Myc antibody.This antibody did not co-precipitate the mutant Ozz protein, confirmingthat Ozz interacts with the Elongin B/C complex through the SOCS domain.

Ozz degradation. It has been suggested that the proteasome is involvedin the degradation of SOCS proteins in hematopoietic cells (Kamura, T.,et al., Genes and Develop., 12:3872-3881, 1998; Narazaki, et al., Proc.Natl. Acad. Sci. USA, 95:13130-13134, 1998). To determine whether thelevels of Ozz protein during muscle differentiation are also modulatedby the proteasome-pathway, primary myoblasts were cultured for 8 hoursin the presence of 10 mM of lactacystin, a specific proteasomeinhibitor. It has been reported that inhibition of the proteasomeresults in the accumulation of large protein aggregates that arerecovered in an insoluble sub-cellular fraction. For this reason, afterincubation with the inhibitor, muscle cell lysates were divided into adetergent-soluble and detergent-insoluble fractions. Ozz was barelydetectable in untreated proliferating myoblasts but accumulated in theinsoluble fraction after inhibition of the proteasome with lactacystin.Thus, in undifferentiated myoblasts Ozz is expressed but itsintracellular concentration is maintained at low levels by a rapidturnover rate controlled by the proteasome. In contrast, in myotubes Ozzis only partially degraded by the proteasome as shown by the slightincrease of Ozz protein in the insoluble fraction of lactacystin-treatedmyotube, compared to untreated cells. These data suggest that thesynthesis and degradation of Ozz is tightly regulated during myogenesisand is dependent on the differentiation-state of the cells.

The intracellular distribution of the accumulated, detergent-insolublepool of Ozz was further assessed by immunofluorescent staining ofmyoblast cultures, treated or untreated with lactacystin. For thispurpose, primary myoblasts were infected with an MSCV-based bicistronicretroviral vector expressing Ozz cDNA and the green fluorescent protein(MSCV-OZZ-IRES-GFP) selectable marker. Infected cells were stainedsimultaneously with anti-Ozz and anti-myosin antibodies, used as markerof myoblast differentiation. Ozz accumulated in a large perinuclearaggregate in undifferentiated myoblasts; the size of this Ozz-positivestructure was greatly reduced in the differentiated myotube. Theperinuclear aggregate containing Ozz showed striking similarity to arecently described novel subcellular compartment, the aggresome(Johnston, et at., J. Cell Biol., 143:1883-1898, 1998). Aggresomeformation is believed to be a general response of cells to stressconditions and occurs when the production of aggregation-prone misfoldedproteins saturates the proteasome capacity or when the proteasome isimpaired.

These results suggest that during myogenesis Ozz undergoespost-translational regulation that affects its stability and results inthe increase of endogenous Ozz after myoblast differentiation.

Proteasome-mediated degradation of Ozz. To ascertain the role of theinteraction between Elongin B/C and Ozz in Ozz-stabilization, weinfected primary myoblasts with the MSCV-OZZ-IRES-GFP retroviral vectoras well as a similar vector virus carrying the deleted Ozz cDNA(MSCV-DOZZ-IRES-GFP). Wild type Ozz is expressed at significantly higherlevels than the mutated Ozz although a comparable amount of GFPexpression was obtained with the two vector. However, the level of DOzzprotein increased considerably in the presence of the proteasomeinhibitor, suggesting that the SOCS-box domain in the wild type proteinis necessary to protect Ozz from the proteasome-induced degradation,possibly through the interaction with the Elongin B/C complex.

Ozz phosphorylation. To identify the signals influencing the stabilityof Ozz during in vitro myogenesis, we investigated the role of theputative tyrosine-phosphorylation site presents at the carboxy-terminusof the Ozz SOCS-box. We first tested whether Tyr284 was phosphorylatedin NIH3T3 cells infected with two MSCV retroviruses carrying either thewild type or the mutated Ozz cDNA, used as control. Indeed thevirus-encoded Ozz was phosphorylated since it could be detected with ananti-phosphotyrosine antibody only in cells infected with the wild typeand not with the DOzz vector. Moreover, in primary myoblast cultures wewere able to demonstrate that the endogenously expressed Ozz istyrosine-phosphorylated only in the undifferentiated state, while inmyotubes Ozz undergoes de-phosphorylation.

Taken together these observations raised the possibility that duringmyogenesis, phosphorylation of Tyr284 present in the SOCS-box may affectthe interaction between Ozz and Elongin B/C, and in turn the stabilityof Ozz.

Ozz effects on intracellular β-catenin levels. It has been demonstratedthat the multi-protein complex composed of Elongin B/C, the vonHippel-Lindau tumor-suppressor protein (pVHL), one of the cullins(Cul-2) and the Rbx-1 protein may function as an E3 ubiquitin-ligasethat address pVHL-bound substrate proteins to proteasome-mediateddegradation (Iwai, et al. Proc. Natl. Acad. Sci. USA, 96:12436-12441,1999). It has also been postulated that the SOCS proteins represent afamily of adapter molecules analogous to the pVHL, which could beinvolved, as pVHL, in targeting specific substrates to the proteasomedegradative pathway. This hypothesis led us to examine whetherperturbing Ozz expression in primary myoblasts may influence thestability of one of the Ozz interacting proteins, i.e. β-catenin. Wefound that in differentiated myotubes the mutant DOzz acts as adominant-negative molecule on the endogenously expressed Ozz. Indeed,exogenous overexpression of DOzz caused an accumulation of β-catenin,although overexpression of wild type Ozz did not significantly influenceβ-catenin levels, probably because the system was already saturated.

Since the expression of mutant DOzz, which is unable to bind the ElonginB/C, perturbs the endogenous β-catenin levels, we can speculate that inthe differentiated myotube Ozz targets its interacting substrateβ-catenin to the proteasome pathway through interaction with the ElonginB/C.

Ozz expression in galactosialidosis Given the genomic organization ofthe Ozz and PPCA genes, mutations affecting PPCA function and causinggalactosialidosis could potentially influence or deregulate expressionof Ozz as well. With this in mind we have tested the intracellulardistribution and level of expression of the Ozz protein in autopsyspecimens of the heart of an early infantile galactosialidosis patientwith severe hypotonia and cardiac involvement. As mentioned earlier,immunostaining of adult mouse heart revealed a distribution of theprotein in confined region of the atrial cardiomyocytes surrounding thenucleus. Using the anti-mouse Ozz antibodies we could confirm thissubcellular distribution in cells from a normal human atrium. Incontrast, cells from the atrium of the galactosialidosis patient showedan abnormal level and localization pattern of Ozz, suggesting that themutation in the PPCA gene of this patient interfered with Ozzexpression.

This finding helps with the interpretation phenotypic abnormalitiesaffecting striated muscles and reported in some but not all patientswith galactosialidosis. Furthermore, we can anticipate that deregulationof Ozz activity in the developing and/or differentiating muscle fibersmay influence cellular homeostasis and in turn result in heart orskeletal muscle myopathies.

Discussion: Role of Ozz in Myogenesis

The crucial events in skeletal muscle differentiation are coordinated bythe expression of muscle regulatory proteins acting in concert with thebasic HLH transcription factors of the MyoD family. These transcriptionfactors activate muscle-specific gene expression via their interactionwith the E-box regulatory elements. The finding that the Ozz promotercontains three E-boxes correlates with its expression pattern inembryonal and adult tissues, and suggests that this protein may becritical in muscle differentiation.

The common sequence of myogenic events starts with the induction of theMyoD cascade (MyoD-myogenin-MRF4), followed by growth arrest, expressionof structural sarcomeric components and finally fusion into myotubes.Myotube formation depends upon high cell density, whereas the expressionof the different muscle-specific proteins can also be detected insubconfluent cell cultures, independently of the cell fusion.

Stable cell-cell interactions must be established betweenfusion-competent myoblasts as a prerequisite for furtherdifferentiation. Specific involvement of cadherins in this event hasbeen suggested since N-cadherin is expressed at high levels in profusionmyoblasts and M-cadherin antibodies specifically inhibit myocyte fusion.

β-catenin is an intrinsic component of adherens junctions because itlinks cadherins via α-catenin to the actin cytoskeleton. However,β-catenin has been placed downstream of the Wnt and Wg signaling pathwayand can translocate to the nucleus, where it transactivates target genestogether with LEF and Tcf4 transcription factors. Variations in thelevel of β-catenin partners in these different cellular events cansignificantly modify or affect β-catenin signaling. On the basis of ourdata, Ozz protein appears to be a partner of β-catenin in muscle cells.Thus, modulation of Ozz activity in this cell system may regulateβ-catenin stability, localization, and/or activity in normal myogenesisor combinations of these. Furthermore, because of β-catenin involvementin malignant transformation, mutations compromising Ozz activity mayresult in pathologic conditions, including impairing muscle function.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, are approximate, andare provided for description.

All patents, patent applications, publications, and other materialscited herein are hereby incorporated herein reference in theirentireties.

1. An isolated Ozz protein.
 2. The Ozz protein of claim 1 which is ahuman Ozz protein.
 3. The Ozz protein of claim 2 which has an amino acidsequence as depicted in SEQ ID NO:4.
 4. The Ozz protein of claim 1 whichis a mouse Ozz protein.
 5. The Ozz protein of claim 3 which has an aminoacid sequence as depicted in SEQ ID NO:2.
 6. The Ozz protein of claim 4which is encoded by a nucleic acid having a sequence as depicted in SEQID NO:1, or by a nucleic acid which is hybridizable under stringentconditions with a nucleic acid having a sequence as depicted in SEQ IDNO:1 or its complement.
 7. A fragment, analog, or derivative of the Ozzprotein of claim 1, which fragment, analog, or derivative has theability to bind a protein selected from the group consisting ofβ-catenin, myosin, c-Nap, and Alix.
 8. A polypeptide fragment of an Ozzprotein, wherein the fragment has a property selected from the groupconsisting of: a) having about 40% sequence identity to a duplicatedneuralized homology repeats (NHRs) of neuralized protein of Drosophila;b) comprising a stretch of about 30 amino acids at the C-terminushomologous to two regions of neuralized proteins; c) comprising an aminoacid sequence selected from the group consisting of SEQ ID NOS:5, 7, 9,and 11; d) comprising an amino acid sequence selected from the groupconsisting of GTRATR (SEQ ID NO:19), GVCFSR (SEQ ID NO:20), GQPEA (SEQID NO: 21), KGLKDFCKY (SEQ ID NO: 22),PSLQTLCRLVIQRSMVHRLAIDGLHLPKELKDFCKYE (SEQ ID NO:23), and SLxxxCxxxI(SEQ ID NO:24); and e) specific binding activity with an anti-Ozzantibody.
 9. An isolated nucleic acid encoding the Ozz protein ofclaim
 1. 10. The nucleic acid of claim 9 which is a cDNA.
 11. Thenucleic acid of claim 9, wherein the Ozz protein is a human Ozz protein.12. The nucleic acid of claim 11 which comprises a nucleotide sequenceas depicted in SEQ ID NO:3.
 13. The nucleic acid of claim 9, wherein theOzz protien is a mouse Ozz protein.
 14. The nucleic acid of claim 13which comprises a nucleotide sequence as depicted in SEQ ID NO:1.
 15. Anvector comprising a nucleic acid encoding a fragment of an Ozz proteinoperatively associated with an expression control sequence, wherein thefragment of an Ozz protein has the ability to bind a protein selectedfrom the group consisting of β-catenin, myosin, c-Nap, and Alix.
 16. Thevector according to claim 15, wherein the fragment of an Ozz protein isa full length Ozz protein.
 17. A host cell transfected with the vectorof claim
 15. 18. A non-human animal transformed with the vector of claim15, wherein the animal expresses an Ozz protein.
 19. A method forproducing Ozz protein comprising isolating Ozz protein produced by thehost cells of claim 17, wherein the host cells have been cultured underconditions that provide for expression of the Ozz protein by the vector.20. An isolated nucleic acid of at least ten bases that hybridizes understringent conditions with a nucleic acid having a nucleotide sequence asdepicted in SEQ ID NO:1 or SEQ ID NO:3, with the proviso that thenucleic acid is not a PPCA exon Ia.
 21. The nucleic acid of claim 20,wherein at least ten nucleotides are from the nucleic acid sequence asdepicted in SEQ ID NO:1 or SEQ ID NO:3.
 22. An isolated Ozzmuscle-specific promoter.
 23. A vector comprising a heterologous geneoperatively associated with the muscle-specific promoter of claim 22.24. An antibody that specifically binds to the Ozz protein of claim 1.25. A method for detecting an Ozz protein comprising detecting bindingof the antibody of claim 24 to a protein in a sample suspected ofcontaining an Ozz protein, wherein the antibody is contacted with thesample under conditions that permit specific binding with any Ozzprotein present in the sample.
 26. A method for detecting expression ofOzz comprising detecting mRNA encoding Ozz in a sample from a cellsuspected of expressing Ozz.
 27. The method according to claim 28wherein mRNA encoding Ozz is detected by hybridization to anOzz-specific nucleic acid.
 28. The method according to claim 27 whereinthe Ozz-specific nucleic acid is Ozz cDNA.
 29. A method for detectingdamage to muscle tissue comprising detecting an increase in the level ofOzz protein in a blood or a blood fraction, wherein the presence of anincrease in the level of Ozz in blood or a blood fraction indicatesdamage to muscle tissue.
 30. The method according to claim 29 whereinthe muscle is the heart.
 31. A method for detecting a disease associatedwith a defect in Ozz expression in a subject, which method comprisesdetecting an abnormal level or localization of Ozz in muscle cells froma subject.
 32. The method according to claim 31, wherein the disease isgalactosialidosis.
 33. The method according to claim 32, wherein themuscle cells are from the atrium of the heart.